The present invention relates generally to ribozymes. and more specifically, to ribozymes which are capable of cleaving Hepatitis C Virus nucleic acids. as well as to methods which utilize such ribozymes.
Hepatitis C Virus (xe2x80x9cHCVxe2x80x9d) is an RNA virus which is responsible for approximately 75% of all cases of non-A, non-B hepatitis. Based upon epidemiologic and serologic survevs. it has been estimated that at least 1% to 2% of the world population is chronically infected with HCV (Davis et al., xe2x80x9cTherapy for Chronic Hepatitis Cxe2x80x9d in Gastroenterology Clinics of North America. pp. 603-613, 1995). In the United States. approximately 150,000 acute cases occur annually, where it is the ninth leading cause of death. Moreover, approximately 50% of the acutely infected individuals go on to develop chronic liver disease, and of these, 25% will develop cirrhosis. In addition, on a worldwide basis, 50% of the cases of hepatocellular carcinoma are correlated with HCV infection.
HCV is a positive-stranded RNA virus that is related to the flaviviridae family. The virus, which was isolated and characterized in 1989 (Choo et al., Science 244:362-364, 1989) has an xcx9c9.5 Kb linear genome that replicates through a double stranded RNA intermediate, mediated by a virally encoded RNA-dependent RNA polymerase. There is no known DNA intermediate in the replication process. Presumably, because its replication is exclusively through RNA polymerases, which lack proofreading activities, variability in the coding sequences is a hallmark of individual HCV isolates.
At present, the only therapy which shows some promise for the treatment acute and chronic cases of HCV is alpha interferon (Fried and Hoofnagle, Seminars in Liver Disease 15(1):82-91, 1995). Treatment with alpha interferon however, particularly for chronic patients, produces only temporary results. In particular, in most studies where patients with chronic HCV infections are treated with alpha interferon, only 20% to 25% maintain a sustained, long-term response (Fried and Hoofnagle, supra). In addition, treatment with alpha interferon can produce a wide array of side effects, including systemic effects (e.g., fatigue, fever, headache, anorexia, weight loss, nausea, vomiting, diarrhea, and hair loss), neurologic and psychological effects, an increased susceptibility to infections, as well as an assortment of autoimmune diseases.
The present invention provides an effective treatment to combat HCV infection, and further provides other, related advantages.
The present invention provides ribozymes useful to treat or prevent Hepatitis C Virus (xe2x80x9cHCVxe2x80x9d) infection or disease in an organism or subject, as well as methods of treating HCV infection or disease. Reagents such as vectors, host cells, DNA molecules coding for these ribozymes useful in methods of treatment and prevention of HCV infection or disease also are provided.
Accordingly, in one aspect the present invention ribozymes are provided which have the ability to inhibit replication, infectivity, or gene expression of a hepatitis C virus. Within certain embodiments, the ribozyme is a hammerhead or hairpin ribozyme. Within other embodiments, the ribozyme cleaves genomic strand RNA (either the positive or negative strand), representative examples of which include the sequences set forth in Table I, below. In other aspects, the present invention also provides nucleic acid molecules encoding such ribozymes where, within certain embodiments the nucleic acid molecule is DNA or cDNA. Within preferred embodiments, the nucleic acid molecule is under the control of a promoter to transcribe the nucleic acid molecule.
In another aspect, the present invention provides host cells containing the ribozymes described herein, vectors comprising a promoter operatively linked to the nucleic acid molecule which encodes the ribozymes described herein, and host cells containing such vectors. Within certain embodiments, the vector is a plasmid, a viral vector, retrotransposon, or a cosmid. Representative examples of promoters include the polIII and CMV promoters.
In a further aspect, the present invention provides methods for producing a ribozyme, the ribozyme being able to inhibit hepatitis C viral infection and replication in a cell, comprising the step of providing a nucleic acid molecule (e.g., DNA) encoding a ribozyme under the transcriptional control of a promoter (e.g., in a vector), and transcribing the nucleic acid molecule to produce the ribozyme. The method may also further comprise purifying the ribozyme so produced. The ribozyme may be produced in vitro, in vivo or ex vivo.
In yet another aspect, the present invention provides methods of interfering with or preventing HCV replication in a cell infected with HCV, comprising the step of introducing into a cell an effective amount of the ribozymes described herein. In one embodiment, such methods comprise introducing into the cell an effective amount of DNA encoding a ribozyme as described herein and transcribing the DNA to produce the ribozyme.
In still a further aspect, the present invention provides methods of preventing hepatitis C viral infection in a cell susceptible to infection with HCV, comprising the step of introducing into the cell an effective amount of a nucleic acid molecule (e.g., DNA) encoding a ribozyme as described herein and transcribing the DNA to produce the ribozyme.
In preferred embodiments, the methods further comprise administering the cell transduced with a retroviral vector to a mammal of the same species as that from which the transduced cell was obtained. In other preferred embodiments, the cell transduced with the retroviral vector has been obtained from the mammal receiving the transduced cell.
The above-described methods, as well as the compositions described herein, may be utilized to treat or prevent HCV infection or disease in a wide variety of warm-blooded animals or mammals, including for example, humans.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety as if each were individually noted for incorporation.